Current Medicinal Chemistry, 2003, 10, 2471-2483
`2471
`Glucagon-Like Peptide-1 Synthetic Analogs: New Therapeutic Agents for
`Use in the Treatment of Diabetes Mellitus
`
`George G. Holz* and Oleg G. Chepurny
`
`New York University School of Medicine, Department of Physiology and Neuroscience, New York, NY, USA
`
`Abstract: Glucagon-like peptide-1-(7-36)-amide (GLP-1) is a potent blood glucose-lowering hormone now
`under investigation for use as a therapeutic agent in the treatment of type 2 (adult onset) diabetes mellitus.
`GLP-1 binds with high affinity to G protein-coupled receptors (GPCRs) located on pancreatic β-cells, and it
`exerts insulinotropic actions that include the stimulation of insulin gene transcription, insulin biosynthesis,
`and insulin secretion. The beneficial therapeutic action of GLP-1 also includes its ability to act as a growth
`factor, stimulating formation of new pancreatic islets (neogenesis) while slowing β-cell death (apoptosis). GLP-
`1 belongs to a large family of structurally-related hormones and neuropeptides that include glucagon, secretin,
`GIP, PACAP, and VIP. Biosynthesis of GLP-1 occurs in the enteroendocrine L-cells of the distal intestine, and
`the release of GLP-1 into the systemic circulation accompanies ingestion of a meal. Although GLP-1 is
`inactivated rapidly by dipeptidyl peptidase IV (DDP-IV), synthetic analogs of GLP-1 exist, and efforts have
`been directed at engineering these peptides so that they are resistant to enzymatic hydrolysis. Additional
`modifications of GLP-1 incorporate fatty acylation and drug affinity complex (DAC) technology to improve
`serum albumin binding, thereby slowing renal clearance of the peptides. NN2211, LY315902, LY307161, and
`CJC-1131 are GLP-1 synthetic analogs that reproduce many of the biological actions of GLP-1, but with a
`prolonged duration of action. AC2993 (Exendin-4) is a naturally occurring peptide isolated from the lizard
`Heloderma, and it acts as a high affinity agonist at the GLP-1 receptor. This review summarizes structural
`features and signal transduction properties of GLP-1 and its cognate β-cell GPCR. The usefulness of synthetic
`GLP-1 analogs as blood glucose-lowering agents is discussed, and the applicability of GLP-1 as a therapeutic
`agent for treatment of type 2 diabetes is highlighted.
`Keywords: GLP-1, diabetes mellitus, insulin secretion.
`
`A. INTRODUCTION
`
`Glucagon-like peptide-1-(7-36)-amide (GLP-1, also
`known as t-GLP-1 or GLIP) is an intestinally-derived
`insulinotropic hormone that has attracted considerable
`attention by virtue of its proven ability to act as a blood
`glucose lowering agent. The efficacy with which GLP-1
`lowers concentrations of blood glucose in type 2 diabetic
`subjects (adult onset diabetes mellitus) has prompted clinical
`investigations whereby the therapeutic value of GLP-1 as an
`antidiabetogenic agent has been substantiated. Earlier studies
`revealed that GLP-1 is an effective stimulator of insulin
`secretion from pancreatic β-cells located in the islets of
`Langerhans. More recently, it has become appreciated that
`GLP-1 also exhibits trophic factor-like properties, acting to
`stimulate β-cell growth and differentiation. These effects of
`GLP-1 are complemented by its ability to suppress appetite
`and to delay gastric emptying. Since GLP-1 is a naturally
`occurring hormone, and because GLP-1 is not reported to
`exert deleterious side effects, new efforts are currently under
`way to develop GLP-1 synthetic analogs that exhibit an
`optimal pharmacokinetic and pharmacodynamic spectrum of
`
`*Address correspondence to this author at the Department of Physiology
`and Neuroscience, MSB 442, New York University School of Medicine,
`550 First Avenue, NY 10016, New York, USA; Tel: 212-263-5434; Fax:
`212-689-9060; E-mail: holzg01@popmail.med.nyu.edu
`
`action commensurate with their use as therapeutic agents.
`The intention of this review is to highlight recent advances
`in this field and to provide an overview of the potential
`usefulness of GLP-1 and its synthetic analogs for treatment
`of type 2 diabetes mellitus. For a detailed discussion of
`previous studies concerning GLP-1, the reader is referred to
`earlier in-depth reviews of this subject matter [1-9].
`
`B. GLP-1 AS A BLOOD GLUCOSE-LOWERING
`AGENT
`
`The blood glucose-lowering hormone GLP-1 possesses
`insulinotropic properties that indicate its usefulness as a
`therapeutic agent in the treatment of diabetes mellitus. When
`administered by intravenous infusion to type 2 diabetic
`subjects, GLP-1 stimulates pancreatic insulin secretion and
`blunts the postprandial hyperglycemic excursion that is
`typically observed following ingestion of a meal [10-12].
`Short term or continuous infusion of GLP-1 restores fasting
`glycemia in type 2 diabetic subjects [13-19], and available
`evidence indicates that GLP-1 remains an effective blood
`glucose lowering agent under conditions in which subjects
`are not responsive to administered sulfonylureas such as
`tolbutamide, glyburide, or glipizide
`[20-22].
`Antidiabetogenic actions of GLP-1 in type 2 diabetic
`subjects are manifest as an improvement of pancreatic β-cell
`function, as measured by a clear restoration of the missing
`
`0929-8673/03 $41.00+.00
`
`© 2003 Bentham Science Publishers Ltd.
`
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`2472 Current Medicinal Chemistry, 2003, Vol. 10, No. 22
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`Holz and Chepurny
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`first phase component of glucose-dependent insulin secretion
`[10,15,19,23]. Also observed is an augmentation of second
`phase insulin secretion [14] and an increased amplitude of
`pulsatile insulin secretion [24,25]. Simultaneously, levels of
`fasting blood glucose are reduced (typically by 4-5 mmol/L)
`[19]. Such beneficial actions of administered GLP-1 may be
`a consequence of its ability to compensate for a major deficit
`of endogenous GLP-1 synthesis, secretion, or metabolism in
`diabetic subjects [26,27]. The long term therapeutic value of
`GLP-1 is evident given that it is reported to reduce
`circulating hemoglobin A1c levels by as much as 1.3% [19].
`From the standpoint of its usefulness as a blood glucose
`lowering agent, an attractive pharmacodynamic property of
`GLP-1 is that it stimulates insulin secretion only under
`conditions in which the concentration of blood glucose is
`elevated. As concentrations of blood glucose fall in response
`to administered GLP-1, the insulin secretagogue action of
`GLP-1 is self-terminating [10]. Therefore, unlike
`administered insulin, a natural safeguard exists whereby
`GLP-1 is less likely to induce hypoglycemia in type 2
`diabetic subjects [28,29]. These antidiabetogenic properties
`of GLP-1 are observed not only with intravenous infusion,
`but also
`following subcutaneous administration
`[19,23,29,30-33,] or oral administration via a buccal tablet
`[34].
`Although not fully understood, there is evidence that the
`blood glucose lowering action of GLP-1 is also attributable,
`at least in part, to its ability to suppress pancreatic glucagon
`secretion [11,12,23,35]. This action of the hormone reduces
`hepatic glucose output and may explain, at least in part, an
`ability of GLP-1 to lower levels of blood glucose in type 1
`
`(juvenile-onset) diabetic subjects [11,36]. Interestingly, the
`ability of GLP-1 to suppress pancreatic glucagon secretion
`also appears to be glucose-dependent. Under conditions of
`hypoglycemia, no suppression is observed [37]. Therefore,
`available evidence suggests that GLP-1 does not impair the
`ability of glucagon to act as a counter regulatory hormone in
`support of hepatic glucose output under conditions of
`hypoglycemia.
`It is important to note that GLP-1 is also an inhibitor of
`gastrointestinal secretion and motility, not only in healthy
`individuals [38], but in type 2 diabetic subjects as well
`[21,31,39]. GLP-1 slows gastric emptying and delays
`nutrient absorption, actions that are likely to play a major
`role in determining the effectiveness of GLP-1 as a regulator
`of blood glucose concentration during the time immediately
`following ingestion of a meal [40]. The inhibition of gastric
`emptying by GLP-1 appears to reflect its normal
`physiological role as an intestinal hormone because new
`evidence exists that it mediates the "ileal brake"
`phenomenon [41]. Concomitant with this gastrointestinal
`action, GLP-1 also acts within the central nervous system as
`a mediator of satiety [42]. Evidence has been presented that
`GLP-1 suppresses appetite [43,44], an effect of the hormone
`that appears to involve hypothalamic appetite control centers
`where GLP-1 is synthesized [45,46] and where GLP-1
`receptors are expressed [46,47].
`Although controversial, there is limited evidence that
`GLP-1 exerts an insulinomimetic action to directly facilitate
`glucose uptake in peripheral tissues such as liver, fat or
`muscle. Early on it was recognized that GLP-1 increases
`glucose disposal in type 1 diabetic subjects [11] and healthy
`
`Fig. (1). (A) Alignment of the secretin-like family of peptide hormones including GLP-1-(7-36)-amide. (B) Precursor GLP-1-(1-37)
`aligned with mature forms of the hormone consisting of GLP-1-(7-37) and GLP-1-(7-36)-amide as well as the immediate degradation
`product GLP-1-(9-36)-amide.
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`Glucagon-Like Peptide-1 Synthetic Analogs
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`Current Medicinal Chemistry, 2003, Vol. 10, No. 22 2473
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`subject [48], while simultaneously reducing the postprandial
`glycemic excursion [35,36,49]. Of particular interest is one
`report that in the OLETF rat model of type 2 diabetes, GLP-
`1 stimulated uptake of 2-deoxy-D-glucose in skeletal muscle
`while exerting no effect on hepatic glucose output [50].
`Therefore, alterations of glucose disposal in response to
`GLP-1 may not be explained simply by a suppression of
`glucagon secretion. This conclusion is supported by a recent
`report in which GLP-1 was shown to increase insulin
`sensitivity in depancreatized dogs [51]. Similarly, GLP-1
`was reported to increase insulin sensitivity and insulin-
`mediated glucose uptake in elderly and obese diabetic
`subjects [52,53]. Despite these observations, no firm
`conclusion can as yet be made concerning the possible
`existence of GLP-1 receptors on hepatocytes, adipocytes, or
`skeletal muscle, and furthermore, such findings have been
`contradicted by several alternative reports [54-58].
`
`C. GLP-1 AND GLP-1 RECEPTOR-MEDIATED
`SIGNAL TRANSDUCTION
`
`In order to appreciate exactly how GLP-1 may exert
`pancreatic and/or extra-pancreatic effects, it is useful to
`review current concepts concerning GLP-1 biosynthesis and
`action. GLP-1 is one member of a large family of
`structurally-related hormones and neuropeptides that include
`glucagon, secretin, glucose-dependent insulinotropic
`polypeptide (GIP), pituitary adenylyl cyclase activating
`polypeptide (PACAP), and vasoactive intestinal polypeptide
`(VIP) (Fig. 1A). GLP-1 is synthesized by enteroendocrine L-
`cells of the distal intestine, and it is released into the
`systemic circulation concomitant with a meal. Secretion is
`stimulated by carbohydrates, fat, hormones, and neural
`reflexes [59-61]. The intestinal L-cells synthesize and
`process proglucagon such that the principal post translational
`end product is GLP-1 rather than glucagon. In vitro analyses
`of GLP-1 biosynthesis have been facilitated by the
`availability of cell lines (GLUTag, NCI-H716) that exhibit
`regulated secretion of the hormone [62-64]. The specificity
`of post translational processing within L-cells distinguishes
`them from pancreatic α-cells where glucagon is the principal
`end product. The immature form of GLP-1 is GLP-1-(1-37),
`and it is thought to be biologically inert (Fig. 1 B) .
`Processing of GLP-1-(1-37) generates the biologically active
`isopeptides GLP-1-(7-37) and GLP-1-(7-36)-amide, with the
`amidated peptide representing the fully mature form of GLP-
`1. Circulating levels of GLP-1 rise to the low picomolar
`range after ingestion of a meal [65], and GLP-1 is rapidly
`inactivated by serum dipeptidyl peptidase IV (DDP-IV; EC
`3.4.14.5) [66-68]. This generates GLP-1-(9-36)-amide, a
`degradation product devoid of agonist activity at the GLP-1
`receptor (Fig. 1B). No evidence exists demonstrating a
`significant difference in pharmacological action when
`comparing GLP-1-(7-37) and GLP-1-(7-36)-amide.
`The GLP-1 receptor cDNA was first identified by use of
`an expression cloning strategy [69]. Subsequently, it was
`established that GLP-1 receptors are expressed in pancreatic
`islets of Langerhans, stomach, lung, heart, kidney, and brain
`[47]. GLP-1 receptors have also been located to vagal
`sensory afferent nerve endings constituting a hepatoportal
`vein glucose sensor [70,71]. This pattern of tissue specific
`
`expression of receptors underlies the ability of GLP-1 to act
`as a stimulus for insulin secretion, to slow gastric emptying,
`and to suppress appetite. The GLP-1 receptor genomic
`sequence identifies it as a member of Class II (Family B)
`heptahelical GPCRs. Receptors of this family are
`structurally-related and recognize GLP-1, glucagon, GIP,
`secretin, PACAP, VIP, calcitonin, parathyroid hormone
`(PTH), and corticotropin releasing factor (CRF).
`A common feature of Class II GPCRs is their ability to
`mediate stimulatory actions of peptide hormones on cAMP
`production, Ca2+ signaling, and exocytosis. This is also true
`for GLP-1 receptors expressed on pancreatic β-cells. By
`stimulating production of cAMP, GLP-1 acts as a modulator
`of the β-cell glucose signaling system (Fig.2). The GLP-1
`receptor is positively coupled to adenylyl cyclase by
`heterotrimeric Gs proteins [72]. Evidence also exists for
`coupling of GLP-1 receptors to Gi and Gq/11 proteins [73].
`GLP-1 receptor occupancy stimulates cAMP production,
`whereas very little evidence exists for a major effect of GLP-
`1 on β-cell inositol phosphate production. PKA is a down
`stream effector of the GLP-1 receptor, and it is activated by
`an increase of [cAMP]i. Evidence exists for the targeting of
`PKA to specific subcellular compartments via A-kinase
`anchoring proteins (AKAPs) [74,75], thereby offering one
`potential explanation for how the cAMP-dependent actions
`of GLP-1 may be spatially restricted within the β-cell. An
`additional target of cAMP action is the newly recognized
`family of cAMP-binding proteins known as cAMP-regulated
`guanine nucleotide exchange factors (cAMPGEFs, also
`referred to as Epac) [76-79]. The cAMPGEFs couple cAMP
`production to the activation of Rap1, a small molecular
`weight G protein. Novel signaling properties of the GLP-1
`receptor include its ability to activate immediate early genes
`(IEGs) [80], to increase the number of insulin receptors on
`insulin-secreting cells [81,82], to stimulate mitogen-
`activated protein kinases
`(p38 MAPK, ERK),
`phosphatidylinositol 3-kinase [83], atypical protein kinase
`C-ζ [84], Ca2+/calmodulin-regulated protein kinase [85],
`protein kinase B (Akt), and hormone-sensitive lipase [86]. It
`seems likely that at least some of these effects result from
`activation of Epac by GLP-1. Available evidence also
`indicates an important action of GLP-1 to regulate β-cell ion
`channel function. GLP-1 has been reported to inhibit ATP-
`sensitive K+ channels (K-ATP) [87-89] and to facilitate the
`opening of voltage-dependent Ca2+ channels (VDCCs) [3].
`Intracellular Ca2+ release channels are also targeted by GLP-
`1 in a cAMP-dependent manner [74,90,91]. They include the
`type-2 isoform of ryanodine receptor (RYR-2) Ca2+ release
`channels located on the endoplasmic reticulum.
`
`D. GLP-1 AS A ββββ-CELL GLUCOSE COMPETENCE
`HORMONE
`
`It is now established that GLP-1 acts a competence
`hormone in support of glucose-dependent insulin secretion
`[1,87,92,93]. Under conditions in which β-cells are
`metabolically compromised, GLP-1 acts to restore the
`sensitivity of these cells to glucose. Since type 2 diabetes
`mellitus is a metabolic disorder in which β-cells lose their
`ability to respond to glucose, the induction of glucose
`competence by GLP-1 may be of major therapeutic
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`2474 Current Medicinal Chemistry, 2003, Vol. 10, No. 22
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`
`Fig. (2). Illustrated is the role GLP-1 plays as a modulator of the β-cell glucose signaling system. Glucose stimulates insulin
`secretion, and GLP-1 potentiates the action of glucose by activating multiple signal transduction pathways important to Ca2+-
`dependent exocytosis. Uptake of glucose is mediated by the type-2 glucose transporter (Glut2) and glucose is converted to glucose-6
`phosphate by glucokinase, a type-IV hexokinase that is rate-limiting for β-cell glucose sensing. Aerobic glycolysis generates
`metabolic coupling factors, one of which is ATP. An increase of the cytosolic ATP/ADP concentration ratio results in the closing of
`ATP-sensitive K+ channels (K-ATP), thereby producing membrane depolarization and activation of Ca2+ influx through voltage-
`sensitive Ca2+ channels (Ca-VS). Ca2+ influx produces an increase of [Ca2+]i and triggers fusion of insulin secretory granules with the
`plasma membrane. Repolarization of the membrane is due, in part, to the Ca2+-dependent activation of K+ channels (K-Ca). GLP-1
`modulates this sequence of events via second messengers (cAMP, Ca2+), protein kinase A (PKA), guanine nucleotide exchange factors
`(Epac), Ca2+-calmodulin-regulated protein kinase (CaMK), hormone sensitive lipase (HSL), and lipid metabolites including free fatty
`acids (FFAs).
`
`importance. Recent studies suggest that glucose competence
`results from the ability of GLP-1 to act as a glucose-
`sensitizer. The central locus for this effect appears to be
`intermediary metabolism where GLP-1 facilitates glucose-
`dependent mitochondrial ATP production [94]. This key
`observation provides a clear explanation for why the insulin
`secretagogue action of GLP-1 in β-cells is entirely glucose-
`dependent. An initiating event for induction of glucose
`competence is likely to be the release of Ca2+ from
`intracellular Ca2+ stores. This is Ca2+-induced Ca2+ release
`(CICR), and it is stimulated by GLP-1. Available evidence
`suggests that endoplasmic reticulum-derived Ca2+ interacts
`with cAMP
`to facilitate mitochondrial oxidative
`phosphorylation. Intramitochondrial targets of Ca2+ include
`Ca2+-sensitive dehydrogenases, whereas the targets of cAMP
`may include mitochondrial PKA and Epac, although this
`remains to be determined. Regardless of the precise signal
`transduction mechanism involved, it is reasonable to
`hypothesize that the induction of β-cell glucose competence
`by GLP-1 might contribute to its ability to lower
`concentrations of blood glucose in type 2 diabetic subjects.
`
`Indeed, the restoration of first phase insulin secretion by
`GLP-1 in diabetic subjects is understandable in view of this
`hormone's ability to augment glucose-dependent ATP
`production, thereby closing β-cell K-ATP channels. This
`action of GLP-1 at K-ATP is analogous to the effect
`produced by sulfonylureas (tolbutamide, glyburide,
`glipizide), however it is unique in that it is entirely
`dependent on metabolism of glucose rather than being the
`consequence of a direct pharmacological inhibition of K-
`ATP.
`
`E. THE INSULIN SECRETAGOGUE ACTION OF
`GLP-1
`
`GLP-1 stimulates pancreatic insulin secretion under
`conditions in which β-cells are exposed to concentrations of
`glucose typical of the postprandial state (> 7.5 mM). Insulin
`secretion occurs in a pulsatile manner, and GLP-1 increases
`the amplitude of each pulse without changing the pulse
`frequency [24,25,95]. These effects of GLP-1 are
`accompanied by increased oscillatory electrical activity of the
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`Glucagon-Like Peptide-1 Synthetic Analogs
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`Current Medicinal Chemistry, 2003, Vol. 10, No. 22 2475
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`islets due to the generation of action potentials [96].
`Evidence exists indicating that GLP-1 facilitates both the
`triggering (first phase) and augmentation (second phase)
`pathways of glucose-dependent insulin secretion [3].
`Stimulatory effects of GLP-1 on insulin secretion appear to
`be mediated primarily by cAMP [3]. New findings indicate
`that GLP-1 and cAMP target insulin granule-associated
`proteins including Epac [76] and Rim2 (77), thereby
`increasing the likelihood that a readily releasable pool of
`secretory granules will undergo exocytosis in response to an
`increase of intracellular Ca2+ concentration. Simultaneously,
`GLP-1 appears to stimulate refilling of the readily releasable
`pool, an effect attributed to the mobilization of a reserve
`pool of secretory granules [97]. Such direct effects of GLP-1
`on the exocytotic secretory apparatus are complimented by
`the ability of GLP-1 to increase β-cell electrical activity,
`thereby facilitating influx of Ca2+ through VDCCs [87].
`Simultaneously, GLP-1 mobilizes Ca2+ from intracellular
`Ca2+ stores via CICR, thereby amplifying the exocytosis
`triggered by Ca2+ influx [98]. The Ca2+ stores mobilized by
`GLP-1 appear to correspond to those that are sensitive to
`ryanodine, caffeine, and thapsigargin [75,98]. Indeed,
`evidence has been presented that β-cell RYR Ca2+ release
`channels are important intermediaries linking GLP-1-
`stimulated cAMP production to Ca2+ mobilization and the
`initiation of Ca2+-dependent insulin granule exocytosis [98].
`
`STIMULATION OF
`F.
`EXPRESSION BY GLP-1
`
`INSULIN GENE
`
`GLP-1 stimulates insulin gene expression by virtue of its
`ability to increase transcription of the insulin gene while
`simultaneously stabilizing preproinsulin mRNA [99-103].
`GLP-1 also increases translational biosynthesis of
`proinsulin. These effects of GLP-1 resemble the previously
`described action of glucose to increase β-cell insulin content.
`The ability of GLP-1 to stimulate insulin gene transcription
`has been studied in isolated islets of Langerhans and in a
`variety of insulinoma cell lines. In general, GLP-1 exerts
`multiple stimulatory influences on insulin gene promoter
`activity, as expected given that it activates more than one
`signal transduction pathway. These signaling pathways
`converge at the promoter to regulate the function of
`
`transcription factors that interact with specific response
`elements (Fig.3). Although a conventional cAMP signaling
`mechanism involving PKA, CREB, and the insulin gene
`cAMP response element (CRE) has been suggested to play a
`significant role in this effect, it now appears more likely that
`actions of GLP-1 at the CRE are PKA-independent. This
`conclusion is based on studies of the INS-1 insulin-secreting
`cell line where it was demonstrated that pharmacological
`inhibitors of PKA (H-89, KT 5720) failed to block
`stimulatory actions of GLP-1 at a luciferase reporter
`incorporating -410 bp of the rat insulin I gene promoter
`(RIP1-Luc). Interestingly, the action of GLP-1 was shown to
`be blocked by the serine/threonine protein kinase inhibitor
`Ro 31-8220, by transfection of INS-1 cells with a dominant
`negative isoform of CREB (A-CREB), or by introduction of
`inactivating mutations at the CRE. On the basis of these
`observations, it was suggested that stimulatory actions of
`GLP-1 at the CRE are mediated by basic region leucine
`zipper (bZIP) transcription factor related in structure to
`CREB, and that the transactivation function of such bZIPs
`might be upregulated by an Ro 31-8220-sensitive MAPK-
`activated kinase such as RSK and/or MSK.
`the
`that
`Evidence has also been presented
`pancreatic/duodenal homeodomain transcription factor PDX-
`1 mediates stimulatory actions of GLP-1 at A-elements of
`the insulin gene promoter. PDX-1 translocates to the nucleus
`in response to GLP-1, an effect mediated by PKA [104].
`Levels of PDX-1 mRNA are increased by GLP-1 [105,106],
`and binding of PDX-1 to A1 elements of the rat insulin I
`and II gene promoters is facilitated. The transactivation
`function of PDX-1 is stimulated by GLP-1 [107], and GLP-
`1 also stimulates a luciferase reporter incorporating synthetic
`multimerized E2/A4/A3 elements of RIP1. Given that the
`A-elements of the insulin gene promoter are established to
`be mediators of glucose insulinotropic action, they appear to
`be a locus at which nutrient metabolism and hormonal
`signal transduction interact.
`Recently, it has been suggested that NFAT transcription
`factors (nuclear factor of activated T-cells) mediate
`stimulatory effects of GLP-1 on insulin gene transcription
`[108]. Three NFAT binding sites have been identified in the
`rat insulin I gene promoter (RIP1), and it was suggested that
`GLP-1 facilitates binding of NFAT to the promoter in a
`
`Fig. (3). Response elements and transcription factors that are targeted by GLP-1 for increased insulin gene transcription. Within the
`first -400 bp of the promoter are found 3 predicted response elements for NFAT (NFAT-RE-1-3). Also present are A4/A3 elements that
`bind the homeodomain transcription factor PDX-1. A non-palindromic cyclic AMP response element (CRE) mediates stimulatory
`effects of GLP-1 via basic region leucine zipper (bZIP) transcription factors related in structure to CREB.
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`cAMP/PKA and Ca2+-dependent manner. Analogous to the
`previously described stimulatory effect of cAMP and Ca2+ at
`the glucagon gene promoter [109], it was suggested that the
`cAMP and Ca2+-dependence of GLP-1 action at RIP1 is
`explained by activation of protein phosphatase 2B (PP2B,
`calcineurin). In this scheme, PP2B-mediated dephosphoryla-
`tion of NFAT leads to its nuclear translocation and
`subsequent stimulation of insulin gene transcription.
`
`G. NEWLY RECOGNIZED GROWTH FACTOR-
`LIKE EFFECTS OF GLP-1
`Substantial evidence now exists that GLP-1 acts as a β-
`cell growth factor with neogenic [106,110], mitogenic
`[83,84,111,112] and anti-apoptotic activities [112-114].
`Administration of GLP-1 or the GLP-1 analog Exendin-4 to
`rats increases β-cell mass in a partial pancreatectomy model
`of type 2 diabetes [110]. This effect has also been observed
`in rats using the streptozotocin model of diabetes [115], the
`Goto-Kakizaki model of genetic diabetes [116], or in
`glucose-intolerant rats of advanced age [117]. Similarly,
`Exendin-4 and the long lasting GLP-1 derivative NN2211
`increase β-cell mass in db/db diabetic mice [112,118]. In
`each of these animal models it has been suggested that the
`increase of β -cell mass results from the increased
`differentiation of β-cells from precursor pancreatic ductal
`stem cells (neogenesis). Surprisingly, the proliferative
`capacity of preexisting β-cells appears to be stimulated,
`thereby explaining the mitogenic action of GLP-1. These
`effects of GLP-1 and Exendin-4 are associated with increased
`expression of PDX-1, and in fact, the stimulation of PDX-1
`expression may be a pivotal event underlying the
`differentiation of ductal stem cells to β-cells [106,117-123].
`Neogenesis in response to GLP-1 or Exendin-4 has been
`studied in vitro using primary cultures of progenitor β-cells.
`Evidence was presented that GLP-1 and Exendin-4 induce
`the differentiation of human pancreatic islet-derived
`progenitor cells into insulin-producing cells [122,124].
`Findings similar to these were also obtained using primary
`cultures of rat pancreatic ductal cells [125]. Recently, it has
`been suggested that GLP-1 acts not only as a growth factor
`in support of β-cell neogenesis, but also as a morphogen for
`embryonic development of the pancreas [124,126]. It may be
`concluded that the in vivo therapeutic actions of GLP-1 may
`include its ability to stimulate pancreatic growth and
`differentiation in human diabetic subjects where β-cell mass
`is insufficient to compensate for insulin resistance.
`The increase of β-cell mass observed in response to long
`term (days) treatment with GLP-1 is preceded by a short
`term (hrs) action of the hormone. This short term effect is
`referred to as "functional maturation" whereby immature β-
`cells become competent to secrete insulin in response to
`glucose. Although functional maturation has been described
`in studies of fetal human and pig islet-like cell clusters
`[127,128], it is not clear at the present time whether this
`phenomenon results from immediate (i.e., within seconds)
`effects of GLP-1 on intermediary metabolism (as is the case
`for the induction of Glucose Competence), or alternatively,
`major alterations in the level of expression of key signaling
`molecules subserving β-cell glucose signaling (Glut2
`transporter, glucokinase, K-ATP channels).
`
`To investigate the mitogenic activity of GLP-1, Buteau
`and co-workers [129] studied INS-1 insulin-secreting cells
`that serve as a model system for analyses of β-cell signal
`transduction. It was reported that the proliferation of INS-1
`cells in response to GLP-1 results from "transactivation" of
`the EGF receptor. In this signaling event, GLP-1 activates c-
`Src and promotes the release of a soluble EGF receptor
`ligand (betacelluline), thereby initiating a cascade of protein
`kinase-mediated phosphorylation reactions catalyzed by PI-
`3K, atypical PKC-ζ, and PKB, among others [112,129].
`These findings are of interest because they suggest a possible
`usefulness of GLP-1 as a stimulus for the in vitro expansion
`of progenitor β-cells prior to induction of differentiation.
`Such a strategy might allow large numbers of β-cells to be
`derived in cell cultures, thereby providing a source of
`insulin-secreting cells for use in transplantation.
`The beneficial action of GLP-1 extends to its ability to
`slow β-cell apoptosis and to protect against stress. For
`example, GLP-1 and Exendin-4 protect against β-cell
`apoptosis resulting from the treatment of mice with
`streptozotocin [114]. Similarly, both GLP-1 and Exendin-4
`protect against cytokine-induced cell death in preparations of
`rat β-cells [114]. The importance of such findings is
`emphasized by a recent report demonstrating that the
`protective action of GLP-1 is evident in Zucker diabetic
`fa/fa rats, a genetic model of obesity-related type 2 diabetes
`[113]. It is also of interest that GLP-1 appears to exert a
`cytoprotective action not only in β-cells, but also in neurons
`[130]. Such findings extend on previous studies
`demonstrating that PACAP, a neuropeptide related in
`structure to GLP-1, exerts a neuroprotective effect within the
`brain [131]. On the basis of these observations, it may be
`speculated that one unexpected and beneficial therapeutic
`action of GLP-1 may be its ability to protect against β-cell
`apoptosis associated with autoimmune destruction (type 1
`diabetes) or β-cell exhaustion (type 2 diabetes).
`In conclusion, the above summarized actions of GLP-1
`are understandable if there exists a compensatory mechanism
`whereby biosynthesis of GLP-1 is stimulated under
`conditions in which β-cells are stressed. Evidence that this is
`in fact the case is provided by one recent report
`demonstrating that the biosynthesis of GLP-1 is stimulated
`in rats made diabetic by treatment with streptozotocin [132].
`In this animal model of diabetes, increased biosynthesis of
`GLP-1 occurs secondary to the increased expression of a
`prohormone converting enzyme (PC1/3) that processes
`proglucagon to generate GLP-1. Evidently, accelerated
`biosynthesis of GLP-1 is an evolutionary adaptation to
`stress, thereby allowing an organism to match intra-islet
`levels of GLP-1 to the prevailing need for β - c e l l
`mitogenesis, neogenesis, and differentiation. Summarized in
`(Fig.4) are the multiple signaling pathways by which GLP-1
`influences growth, differentiation, neogenesis, survival,
`insulin biosynthesis, and insulin secretion in β-cells.
`
`H. GLP-1 SYNTHETIC ANALOGS OBTAINED BY
`CHEMICAL MODIFICATION
`
`Clinical studies have demonstrated that the blood
`glucose lowering action of GLP-1 is transient owing to the
`short plasma half life (1.5-5.0 min) of the peptide. Sustained
`
`MPI EXHIBIT 1031 PAGE 6
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`MPI EXHIBIT 1031 PAGE 6
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`

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`Glucagon-Like Peptide-1 Synthetic Analogs
`
`Current Medicinal Chemistry, 2003, Vol. 10, No. 22 2477
`
`Fig. (4). Summary of the biological actions of GLP-1 in β-cells. Illustrated are the second messengers, kinases, and transcription
`factors that mediate insulinotropic effects of GLP-1 on insulin biosynthesis and secretion. Also illustrated are the growth factor-like
`actions of GLP-1 that are important to proliferation, neogenesis, differentiation, and survival of β-cells.
`
`lowering of blood glucose concentration is only observed
`with continuous infusion, as demonstrated in studies in
`which GLP-1 was administered by intravenous infusion over
`a 24 hr time course [22]. Clearly, this mode of
`administration is not compatible with general use by the
`public. To address this short coming, efforts have been
`directed to engineer long lasting analogs of GLP-1 that are
`synthetic peptides and which exhibit insulinotropic and
`blood glucose lowering activities in vivo. These peptides
`include NN221